Findings to date suggest three types of methods for expressing and accumulating high levels of exogenous gene products in rice endosperm tissues while avoiding degradation during the ripening process. In the first of these methods that are now actually in use, the desired protein or peptide is introduced into the variable region of a seed storage protein of rice, or some other species, for translocation into vacuole-derived type II protein bodies (PB-IIs) as a part of that storage protein. The second method is useful in cases where the desired protein does not originally comprise a vacuolar translocation signal. It involves attaching a signal peptide to the N terminus of the desired protein and secreting it to the cell wall apoplast. In the third method, a signal peptide is attached to the N terminus of the desired protein and a known endoplasmic reticulum localization signal, such as KDEL (SEQ ID NO: 17) or HDEL (SEQ ID NO: 18), is attached to the C terminus; the desired protein is then accumulated in the endoplasmic-reticulum.
Rice endosperm comprises PB-Is, protein bodies which are not vacuole-derived and in which primarily prolamines accumulate. Since PB-Is are produced directly from the endoplasmic reticulum, their production process is vastly different from that of vacuole-derived PB-II protein bodies. With regards to the accumulation of prolamines in PB-Is, a Washington State University group reported that prolamine mRNAs are transported to PB-Is via their 3′ untranslated region and translated therein into prolamines (Choi et al. Nature 2000, 407: 765-767).